Somatic cell reprogramming

ABSTRACT

The present invention relates to methods for reprogramming a somatic cell to pluripotency by administering into the somatic cell at least one or a plurality of potency-determining factors. The invention also relates to pluripotent cell populations obtained using a reprogramming method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/053,440, filed Mar. 21, 2008, which claims the benefit of U.S.Provisional Patent Application No. 60/919,687, filed Mar. 23, 2007; U.S.Provisional Patent Application No. 60/974,980, filed Sep. 25, 2007; andU.S. Provisional Patent Application No. 60/989,058, filed Nov. 19, 2007,each of which is incorporated herein by reference as if set forth in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND

Embryonic stem (ES) cells can grow indefinitely while maintainingpluripotency and can differentiate into cells of all three germ layers(Evans & Kaufman, Nature 292:154-156 (1981)). Human ES cells will beuseful in treating a host of diseases, such as Parkinson's disease,spinal cord injury and diabetes (Thomson et al., Science 282:1145-1147(1998)). Scientists have sought technical solutions to avoid the currentmethod of generating ES cells from blastocyst cells and to avoidanticipated tissue rejection problems following transplantation intopatients. One desirable way to accomplish these solutions would be togenerate pluripotent cells directly from somatic cells of a post-natalindividual.

Somatic cells can be reprogrammed by transferring their nuclear contentsinto oocytes (Wilmut et al., Nature 385:810-813 (1997)) or by fusionwith ES cells (Cowan et al., Science 309:1369-1373 (2005)), indicatingthat unfertilized eggs and ES cells contain factors that confertotipotency or pluripotency in somatic cells.

Likewise, Yu et al. showed that cells derived by in vitrodifferentiation from an H1 Oct4 knock-in ES cells did not express EGFP,but that EGFP expression was restored upon cell-cell fusion with humanES cells. Yu et al., Stem Cells 24:168-176 (2006), incorporated hereinby reference as if set forth in its entirety). Therefore, Yu et al.demonstrated that differentiated cells can become pluripotent viacell-cell fusion with human ES cells. Regardless of the differentiatedcell type, upon fusion with undifferentiated human ES cells, ES cellspecific antigens and marker genes were expressed anddifferentiation-specific antigens were no longer detectable in the fusedhybrid cells. Advantageously, EGFP expression was re-established in thehybrid cells, providing a convenient marker for re-establishment ofpluripotent stem cell status. When the hybrid cells formed embryoidbodies (EBs), genes characteristic of all three germ layers andextra-embryonic tissues were up-regulated, indicating that the hybridcells had a potential to differentiate into multiple lineages.

Although the transcriptional determination of pluripotency is not fullyunderstood, several transcription factors, including Oct 3/4 (Nichols etal., Cell 95:379-391 (1998)), Sox2 (Avilion et al., Genes Dev.17:126-140 (2003)) and Nanog (Chambers et al., Cell 113:643-655 (2003))are involved in maintaining ES cell pluripotency; however, none issufficient alone to specify ES cell identity.

Chambers & Smith (EP 1 698 639 A2, (2002)) maintained pluripotent murinecells without a feeder layer or feeder cell extract and without a gp130cytokine by introducing vectors that encode or activatedifferentiation-suppressing factors, but did not convert differentiatedcells into a pluripotent state.

More recently, Takahashi & Yamanaka introduced four factors (i.e.,Oct3/4, Sox2, c-Myc and Klf4) into mouse ES cells and mouse adultfibroblasts cultured under conditions suitable for mouse ES cell cultureto obtain induced pluripotent stem (iPS) cells that exhibited mouse EScell morphology and growth properties and expressed mouse ES cell markergenes (Takahashi & Yamanaka, Cell 126:663-676 (2006)). Notably,exogenous Oct-4 introduced into the mouse fibroblasts resulted in onlymarginal Oct-4 expression. Subcutaneous transplantation of iPS cellsinto nude mice resulted in tumors containing a variety of tissues fromall three germ layers. Following injection into blastocysts, iPS cellscontributed to mouse embryonic development. However, c-Myc, which wasnecessary for pluripotent induction, is an oncogene. Likewise, Klf4 isan oncogene. These data demonstrate that pluripotent cells can bedirectly generated from mouse fibroblast cultures by adding only a fewdefined factors using a retroviral transduction. However, as describedinfra, the set of factors used to produce iPS cells from differentiatedmouse cells was insufficient to reprogram human somatic cells topluripotency using lentiviral vectors without introducing additionalchanges to the cells.

One could hypothesize that factors that can reprogram human somaticcells differ from those factors that can reprogram somatic cells frommodel organisms (including mice) because ES cells from mice and humansrequire distinct sets of factors to remain undifferentiated,illustrating the significance of species-specific differences, evenamong mammals. For example, the leukemia inhibitory factor (LIF)/Stat3pathway, a key to mouse ES cell proliferation, does not support human EScell proliferation and appears inactive in conditions that support humanES cells (Daheron L, et al., Stem Cells 22:770-778 (2004); Humphrey R,et al., Stem Cells 22:522-530 (2004); and Matsuda T, et al., EMBO J.18:4261-4269 (1999)).

Similarly, while bone morphogenetic proteins (BMPs) together with LIFsupport mouse ES cell self-renewal at clonal densities in serum-freemedium (Ying Q, et al., Cell 115:281-292 (2003)), they cause rapid humanES cell differentiation in conditions that would otherwise supportself-renewal, such as culture on fibroblasts or infibroblast-conditioned medium (Xu R, et al., Nat. Biotechnol.20:1261-1264 (2002)). Indeed, inhibition of BMP signaling in human EScells is beneficial (Xu R, et al., Nat. Methods 2:185-190 (2005)).

Still further, fibroblast growth factor (FGF) signaling is important toself-renewal of human ES cells, but apparently not for mice (Xu et al.,(2005), supra; and Xu C, et al., Stem Cells 23:315-323 (2005)).

Accordingly, the art still seeks a set of potency-determining factorssuited at least for use in methods for reprogramming primate (includinghuman and non-human) somatic cells to yield pluripotent cells. Suchcells, obtained without relying upon embryonic tissues, would be suitedfor use in applications already contemplated for existing, pluripotent,primate ES cells.

BRIEF SUMMARY

The present invention is broadly summarized as relating to methods forreprogramming differentiated, somatic, primate cells into pluripotentcells, and more specifically into iPS cells. As used herein, “iPS cells”refer to cells that are substantially genetically identical to theirrespective differentiated somatic cell of origin and displaycharacteristics similar to higher potency cells, such as ES cells, asdescribed herein. The cells can be obtained from various differentiated(i.e., non-pluripotent and multipotent) somatic cells.

iPS cells exhibit morphological (i.e., round shape, large nucleoli andscant cytoplasm) and growth properties (i.e., doubling time; ES cellshave a doubling time of about seventeen to eighteen hours) akin to EScells. In addition, iPS cells express pluripotent cell-specific markers(e.g., Oct-4, SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, but not SSEA-1). iPScells, however, are not immediately derived from embryos and cantransiently or stably express one or more copies of selectedpotency-determining factors at least until they become pluripotent. Asused herein, “not immediately derived from embryos” means that thestarting cell type for producing iPS cells is a non-pluripotent cell,such as a multipotent cell or terminally differentiated cell, such assomatic cells obtained from a post-natal individual.

In the methods described herein, at least two potency-determiningfactors can be introduced into, and expressed in, differentiated somaticcells, whereupon the somatic cells convert in culture to cells havingproperties characteristic of pluripotent cells, such as human ES cells(i.e., express at least Oct-4, SSEA-3, SSEA-4, TRA-1-60 or TRA-1-81, butnot SSEA-1, and appear as compact colonies having a high nucleus tocytoplasm ratio and prominent nucleolus), that can differentiate intocells characteristic of all three germ layers, and that contain thegenetic complement of the somatic cells of a post-natal individual.Apart from genetic material introduced to encode the potency-determiningfactors, the reprogrammed (i.e., converted) cells are substantiallygenetically identical to the somatic cells from which they were derived.

As used herein, a “potency-determining factor” refers to a factor, suchas a gene or other nucleic acid, or a functional fragment thereof, aswell as an encoded factor or functional fragment thereof, used toincrease the potency of a somatic cell, so that it becomes pluripotent.The potency-determining factors optionally can be present onlytransiently in the reprogrammed cells or can be maintained in atranscriptionally active or inactive state in the genome of thereprogrammed cells. Likewise, the potency-determining factors can bepresent in more than one copy in the reprogrammed cells, where thepotency-determining factor can be integrated in the cell's genome, canbe extra-chromosomal or both. The potency-determining factors caninclude, but are not limited to, Stella (SEQ ID NO:1); POU5F1 (Oct-4;SEQ ID NO:2), Sox2 (SEQ ID NO:3), FoxD3, UTF1, Rex1, ZNF206, Sox15,Myb12, Lin28 (SEQ ID NO:4), Nanog (SEQ ID NO:5), DPPA2, ESG1, Otx2 andsubsets thereof. In some embodiments, as few as two potency-determiningfactors, e.g., Oct-4 and Sox2, can be sufficient. Efficiency inobtaining reprogrammed cells, however, can be improved by includingadditional potency-determining factor, such as Lin28, Nanog or both.

In a first aspect, the invention relates to a replenishable, enrichedpopulation of pluripotent cells obtained from a post-natal individual,especially from a living individual, but optionally from a deceasedindividual. Cells in the enriched cell population express at least onecell-type-specific marker, including, but not limited to, Oct-4, SSEA3,SSEA4, Tra-1-60, Tra-1-81 or combinations thereof and have otherhallmarks of pluripotent cells, such as ES cells. In addition, thepluripotent cells may express alkaline phosphatase (ALP). Furthermore,the pluripotent cells may have a genome substantially geneticallyidentical to that of a pre-existing, differentiated cell from theindividual. Likewise, the pluripotent cells may have a genome thatencodes at least one of the potency-determining factors, which may betranscriptionally active or inactive after reprogramming. Additionally,the potency-determining factors may be in a form of a reprogrammingsequence in which a polynucleotide encoding the potency-determiningfactor is operably linked to a heterologous promoter. As used herein,“heterologous promoter” means a promoter that is operably linked to apolynucleotide for which the promoter does not normally initiatetranscription.

In a second aspect, the invention relates to methods and compositionsfor identifying potency-determining factors required to reprogramsomatic cells into pluripotent cells.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although suitable methods andmaterials for the practice or testing of the present invention aredescribed below, other methods and materials similar or equivalent tothose described herein, which are well known in the art, can also beused.

Other objects, advantages and features of the present invention willbecome apparent from the following specification taken in conjunctionwith the accompanying drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 illustrates a site downstream from a human Oct4 promoter intowhich a knock-in construct was introduced. In cells containing theknock-in construct, enhanced green fluorescent protein (EGFP) andneomycin phosphotransferase (NEO) are expressed when the Oct4 promoteris active. These cells can be used to evaluate which factors canreprogram somatic cells into pluripotent cells.

FIGS. 2A-B illustrate human H1 ES cell differentiation. FIG. 2A showsschematics of myeloid precursor derivation and purification from humanES cells. FIG. 2B shows phenotypic analysis of differentiated cellsobtained after Percoll® separation. Gray line: isotype control; blackline: antibody staining Abbreviations: hESC, human embryonic stem cell;MPO, myeloperoxidase; pHEMA, poly(2-hydroxyethyl methacrylate).

FIG. 3 illustrates the Oct-4 region containing the knock-in construct ofFIG. 1.

FIGS. 4A-C illustrate lentiviral transduction of somatic cells. FIG. 4Ashows a schematic diagram of lentiviral construct. FIG. 4B showsPercoll®-purified cells were transduced with EGFP-expressing lentiviralvectors at various MOI. EGFP expression was analyzed by flow cytometrythree days after transduction without drug selection. FIG. 4C showslentiviral transduction of Percoll®-purified cells after severaladditional days of culture on Matrigel®. EGFP expression was analyzedtwo days after lentiviral transduction.

FIG. 5 illustrates transgene overexpression in cells differentiated forseven days on Matrigel®. No significant change in morphology wasobserved in cells overexpressing Nanog or EGFP (control). Morphology ofOct-4-expressing cells changes dramatically, and many of these cellssurvived neomycin selection, but none of these cells showed typicalhuman ES cell morphology, indicating that a drug-selectable populationof Oct-4-expressing ES cells does not persist through the culture periodnecessary for myeloid differentiation.

FIGS. 6A-B illustrate reprogramming of Oct4KICD45+A cells throughintroduction of fourteen potency-determining factors. FIG. 6A shows theestablished clones display undifferentiated human ES cell morphology andexpress EGFP under direction of the endogenous Oct4 promoter. FIG. 6Bshows flow cytometry analysis of human ES cell-specific cell surfaceantigen expression in established clones. Gray line: isotype control;black line: antibody staining.

FIGS. 7A-C illustrate reprogramming efficiency, as evidenced by colonyformation, after introduction of various sets of potency-determiningfactors. FIG. 7A shows the identified set of fourteenpotency-determining factors was introduced into cells in combinations,wherein each combination excluded one of the fourteen factors. Byevaluating the ability of the potency-determining factors to reprogramthe tested cells to an ES-like state, the inventors determined whetherthe excluded potency-determining factor was essential to thereprogramming. For example, a set of potency-determining factors termedM1 that lacked Oct-4 (depicted as M1—Oct-4) was unable to form asignificant number of ES-like colonies. As such, it was concluded thatOct-4 was important for somatic cell reprogramming. FIG. 7B shows a setof potency-determining factors (narrowed from FIG. 7A) evaluated forfurther testing was narrowed from fourteen to four (M4, being Oct-4,Sox2, Lin28 and Nanog). These four potency-determining factors weretested by serially excluding one of the four from the combination. Wherea combination of three potency-determining factors (e.g., M4 Oct. 4) wasunable to reprogram the tested cells to form a significant number ofstable ES-like colonies, the inventors concluded that the omitted geneis important for somatic cell reprogramming. In FIG. 7B, the light graybars indicate the total number of reprogrammed colonies formed havingtypical human ES cell morphology; dark gray bars indicate the number oflarge colonies with minimal differentiation. FIG. 7C shows a set ofpotency-determining factors (narrowed from FIG. 7B) evaluated forfurther testing was narrowed from four to two (i.e., Oct-4 and Sox2).Oct-4, Sox2, Lin28 and Nanog were tested by serially excluding two ofthe four from the combination.

FIGS. 8A-B illustrate reprogramming in human adult skin fibroblasts.FIG. 8A shows bright-field images of human adult skin cell (p5) (left)and reprogrammed cells (right). FIG. 8B shows flow cytometry analysis ofhuman ES cell-specific markers in human adult skin cells (p5) (bottom)and reprogrammed cells (top). Gray line: isotype control; black line:antibody staining.

FIGS. 9A-B illustrate the effect on reprogramming of relative expressionof Oct-4 and Sox2. FIG. 9A shows Western blot analysis of Oct-4 and Sox2in 293FT cells; lane 1, pSin4-EF2-Oct4-IRES1-Sox2 (OS-IRES1); lane 2,pSin4-EF2-Oct4-IRES2-Sox2 (OS-IRES2); lane 3, pSin4-EF2-Oct4-F2A-Sox2(OS-F2A); lane 4, pSin4-EF2-Oct4-IRES1-puro (O); lane 5,pSin4-EF2-Sox2-IRES1-puro (S); lane 6, no plasmid (control). FIG. 9Bshows reprogramming in mesenchymal cells derived from OCT4 knock-inhuman ES cells using linked potency-determining factors; genecombinations are the same as in FIG. 9A, with the addition ofpSin4-EF2-Nanog-IRES1-puro (N) and pSin4-EF2-Lin28-IRES1-puro (L).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The inventors hypothesized that potency-determining factors present inprimate ES cells play an important role in maintaining pluripotency andthat differentiated somatic cells could be reprogrammed to a state ofpluripotency through expression of potency-determining factors.

Cell types pass through various levels of potency duringdifferentiation, such as totipotency, pluripotency and multipotency. Ofparticular interest herein are pluripotent cells. As used herein,“pluripotent cells” refer to a population of cells that candifferentiate into all three germ layers (e.g., endoderm, mesoderm andectoderm). Pluripotent cells express a variety of pluripotentcell-specific markers, have a cell morphology characteristic ofundifferentiated cells (i.e., compact colony, high nucleus to cytoplasmratio and prominent nucleolus) and form teratomas when introduced intoan immunocompromised animal, such as a SCID mouse. The teratomastypically contain cells or tissues characteristic of all three germlayers. One of ordinary skill in the art can assess thesecharacteristics by using techniques commonly used in the art. See, e.g.,Thomson et al., supra. Pluripotent cells are capable of bothproliferation in cell culture and differentiation towards a variety oflineage-restricted cell populations that exhibit multipotent properties.Multipotent somatic cells are more differentiated relative topluripotent cells, but are not terminally differentiated. Pluripotentcells therefore have a higher potency than multipotent cells. As usedherein, “reprogrammed pluripotent primate stem cells” (and similarreferences) refer to the pluripotent products of somatic cellreprogramming methods. Such cells are suitable for use in research andtherapeutic applications currently envisioned for human ES cells.

The present invention broadly relates to novel methods for reprogrammingdifferentiated somatic cells into higher-potency cells, such aspluripotent cells, by administering at least two potency-determiningfactors into somatic cells to achieve a higher level of potency in thereprogrammed cells than in the somatic cells. Advantageously, thepresent invention allows the generation of pluripotent cells, such asiPS cells, from somatic cells without requiring an addition of cellsurface receptors for introducing the potency-determining factors to thesomatic cells. As used herein, “reprogramming” refers to a geneticprocess whereby differentiated somatic cells are converted intode-differentiated, pluripotent cells, and thus have a greaterpluripotency potential than the cells from which they were derived. Thatis, the reprogrammed cells express at least one of the followingpluripotent cell-specific markers: SSEA-3, SSEA-4, TRA-1-60 or TRA 1-81.Preferably, the reprogrammed cells express all these markers.

Potency-determining factors that can reprogram somatic cells include,but are not limited to, factors such as Oct-4, Sox2, FoxD3, UTF1,Stella, Rex1, ZNF206, Sox15, Myb12, Lin28, Nanog, DPPA2, ESG1, Otx2 orcombinations thereof. In the examples, a set with as few as two of thefourteen factors was sufficient to reprogram the tested cells; this setincluded Oct-4 and Sox2. Addition of other potency-determining factorsto Oct-4 and Sox2, however, increased the efficiency with whichreprogrammed cells were obtained. c-Myc and Klf4, however, are notessential as potency-determining factors. Preferably, thepotency-determining factor may be a transcription factor.

Suitable somatic cells can be any somatic cell, although higherreprogramming frequencies are observed when the starting somatic cellshave a doubling time about twenty-four hours. Somatic cells useful inthe invention are non-embryonic cells obtained from a fetal, newborn,juvenile or adult primate, including a human. Examples of somatic cellsthat can be used with the methods described herein include, but are notlimited to, bone marrow cells, epithelial cells, fibroblast cells,hematopoietic cells, hepatic cells, intestinal cells, mesenchymal cells,myeloid precursor cells and spleen cells. Another type of somatic cellis a CD29⁺ CD44⁺ CD166⁺ CD105⁺ CD73⁺ and CD31⁻ mesenchymal cell thatattaches to a substrate. Alternatively, the somatic cells can be cellsthat can themselves proliferate and differentiate into other types ofcells, including blood stem cells, muscle/bone stem cells, brain stemcells and liver stem cells. Multipotent hematopoietic cells, suitablymyeloid precursor or mesenchymal cells, are specifically contemplated assuited for use in the methods of the invention.

Likewise, suitable somatic cells are receptive, or can be made receptiveusing methods generally known in the scientific literature, to uptake ofpotency-determining factors including genetic material encoding thefactors. Uptake-enhancing methods can vary depending on the cell typeand expression system. Exemplary conditions used to prepare receptivesomatic cells having suitable transduction efficiency are known in theart and are described in the examples below. One method for making cellsreceptive to potency-determining factors is described below inconnection with the electroporation methods.

A potency-determining factor may be introduced as a reprogrammingsequence in which a polynucleotide sequence encoding thepotency-determining factor is operably linked to a heterologouspromoter, which may become inactive after somatic cells arereprogrammed. The heterologous promoter is any promoter sequence thatcan drive expression of a polynucleotide sequence encoding thepotency-determining factor in the somatic cell, such as, e.g., an Oct4promoter.

The relative ratio of potency-determining factors may be adjusted toincrease reprogramming efficiency. For example, linking Oct-4 and Sox2in a 1:1 ratio on a single vector increased reprogramming efficiency incells by a factor of four (FIG. 9A-B) when compared to reprogrammingefficiency wherein the potency-determining factors were provided tocells in separate constructs and vectors, where the uptake ratio of therespective potency-determining factors into single cells wasuncontrolled. Although the ratio of potency-determining factors maydiffer depending upon the set of potency-determining factors used, oneof ordinary skill in possession of this disclosure can readily determinean optimal ratio of potency-determining factors.

Pluripotent cells can be cultured in any medium used to support growthof pluripotent cells. Typical culture medium includes, but is notlimited to, a defined medium, such as TeSR™ (StemCell Technologies,Inc.; Vancouver, Canada), mTeSR™ (StemCell Technologies, Inc.) andStemLine® serum-free medium (Sigma; St. Louis, Mo.), as well asconditioned medium, such as mouse embryonic fibroblast (MEF)-conditionedmedium. As used herein, a “defined medium” refers to a biochemicallydefined formulation comprised solely of biochemically-definedconstituents. A defined medium may also include solely constituentshaving known chemical compositions. A defined medium may further includeconstituents derived from known sources. As used herein, “conditionedmedium” refers to a growth medium that is further supplemented withsoluble factors from cells cultured in the medium. Alternatively, cellscan be maintained on MEFs in culture medium.

The inventors used a serial analysis of gene expression (SAGE) libraryto obtain transcriptome profiles of genes abundant in ES cells.Specifically, a SAGE library was used to identify potency-determiningfactors that regulate pluripotency and self-renewal in ES cells. SAGElibraries are well-known to one of ordinary skill in the art and arepublicly available or can be specifically constructed by companies, suchas Agencourt Bioscience Corp. (Beverly, Mass.).

In another aspect, the invention provides an enriched population ofpluripotent cells substantially genetically identical to cells of apost-natal individual. The cells can be obtained by reprogrammingsomatic cells isolated from the post-natal individual. In someembodiments, the cell population is a purified population, representingat least 60%, 70%, 80% and advantageously greater than 95% of the cellsin the population, and any and all whole or partial integerstherebetween. The reprogrammed cells are euploid, exhibit cellmorphology characteristic of pluripotent cells and express pluripotentcell-specific markers, such as, e.g., Oct-4, SSEA-3, SSEA-4, Tra-1-60,Tra-1-81 or combinations thereof, and form teratomas when introducedinto an immunocompromised animal.

Yet another aspect provides methods and compositions for identifying andusing potency-determining factors sufficient to reprogram somatic cellsinto pluripotent cells. As noted herein, the reprogrammed pluripotentcells contain the genetic complement of, and are substantiallygenetically identical to somatic cells obtained from a post-natalindividual. Generally, methods for identifying potency-determiningfactors include the steps of introducing genetic material encoding oneor a plurality of putative potency-determining factors into somaticcells receptive to uptake of the genetic material under conditionseffective to express the factors encoded on the introduced geneticmaterial at levels sufficient to reprogram the cells to a lessdifferentiated, higher-potency state; and observing a population ofpluripotent cells after introduction of the genetic material. Thepluripotent cells can be characterized by cell morphology, pluripotentcell-specific markers or both. Advantageously, the pluripotent cells canbe identified by expression in the treated cells of a marker provided inthe cells so as to be expressed only upon reprogramming of the cells toa pluripotent state. Through this approach, potency-determining factorscapable of reprogramming somatic cells into pluripotent cells can beidentified, as is described in the examples below.

Genetic material encoding a potency-determining factor can be introducedby transfection or transduction into the somatic cells using a vector,such as an integrating- or non-integrating vector. Of particularinterest herein are retroviral vectors. Retroviral vectors, particularlylentiviral vectors, are transduced by packaging the vectors into virionsprior to contact with a cell. After introduction, the DNA segment(s)encoding the potency-determining factor(s) can be locatedextra-chromosomally (e.g., on an episomal plasmid) or stably integratedinto cellular chromosome(s).

A viral-based gene transfer and expression vector is a genetic constructthat enables efficient and robust delivery of genetic material to mostcell types, including non-dividing and hard-to-transfect cells (primary,blood, stem cells) in vitro or in vivo. Viral-based constructsintegrated into genomic DNA result in high expression levels. Inaddition to a DNA segment that encodes a potency-determining factor ofinterest, the vectors include a transcription promoter and apolyadenylation signal operatively linked, upstream and downstream,respectively, to the DNA segment. The vector can include a single DNAsegment encoding a single potency-determining factor or a plurality ofpotency-determining factor-encoding DNA segments. A plurality of vectorscan be introduced into a single somatic cell. The vector can optionallyencode a selectable marker to identify cells that have taken up andexpress the vector. As an example, when the vector confers antibioticresistance on the cells, antibiotic can be added to the culture mediumto identify successful introduction of the vector into the cells.Integrating vectors can be employed, as in the examples, to demonstrateproof of concept. Retroviral (e.g., lentiviral) vectors are integratingvectors; however, non-integrating vectors can also be used. Such vectorscan be lost from cells by dilution after reprogramming, as desired. Asuitable non-integrating vector is an Epstein-Barr virus (EBV) vector.Ren C, et al., Acta. Biochim. Biophys. Sin. 37:68-73 (2005); and Ren C,et al., Stem Cells 24:1338-1347 (2006), each of which is incorporatedherein by reference as if set forth in its entirety.

The vectors described herein can be constructed and engineered usingart-recognized techniques to increase their safety for use in therapyand to include suitable expression elements and therapeutic genes.Standard techniques for the construction of expression vectors suitablefor use in the present invention are well-known to one of ordinary skillin the art and can be found in such publications such as Sambrook J, etal., “Molecular cloning: a laboratory manual,” (3rd ed. Cold SpringHarbor Press, Cold Spring Harbor, N.Y. 2001), incorporated herein byreference as if set forth in its entirety.

The ability to identify and enrich for pluripotent cells can befacilitated by providing a non-lethal marker in the somatic cells, suchas Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein(EGFP) or luciferase, under the control of a promoter active only afterthe somatic cell has converted to a pluripotent state. A selectablemarker gene is used to identify the reprogrammed cells expressing themarker through visible cell selection techniques, such as fluorescentcell sorting techniques. Alternatively, the reprogrammed cells can beproduced without a selectable marker. In the examples below, a markerwas provided in the genome of the somatic cells downstream of thepromoter that regulates Oct-4 expression. The endogenous Oct4 promoteris active in undifferentiated, pluripotent ES cells. A drug-selectablepopulation of Oct-4-expressing ES cells did not persist through theculture period necessary for myeloid differentiation. However, becausesome Oct-4 expression can persist into early stages of differentiation,it is appropriate to enrich the population for pluripotent cells byselecting colonies having characteristic ES cell morphology and bymaintaining the cells under ES cell maintenance culture conditions. Itis not intended that all cells in the reprogrammed cell culture have thedesired level of potency. Given the inefficiencies of cell sortingtechnology, the variations in levels of gene expression and otherbiological effects, some cells in the enriched population may not bepluripotent. However, at a practical level, the reprogrammed cellpopulation derived from somatic cells is enriched for pluripotent cells.

The non-lethal marker can be constructed to enable its subsequentremoval using any of a variety of art-recognized techniques, such asremoval via Cre-mediated, site-specific gene excision. For example, itmay become desirable to delete the marker gene after the pluripotentcell population is obtained, to avoid interference by the marker geneproduct in the experiment or process to be performed with the cells.Targeted deletions can be accomplished by providing structure(s) nearthe marker gene that permits its ready excision. That is, a Cre/Loxgenetic element can be used. The Lox sites can be built into the cells.If it is desired to remove the marker from the pluripotent cells, theCre agent can be added to the cells. Other similar systems also can beused. Because Cre/Lox excision can introduce undesirable chromosomalrearrangements and can leave residual genetic material after excision,the inventors recognize the desirability of introducing thepotency-determining factors into the somatic cells usingnon-integrating, episomal vectors and obtaining cells from which theepisomal vectors are lost (e.g., at a rate of about 5% per generation)by subsequently withdrawing the drug selection used to maintain thevectors during the reprogramming step.

The following examples are provided as further non-limitingillustrations of methods for identifying potency-determining genes orfactors for converting somatic cells into pluripotent cells. In someexamples, human H1 Oct4 knock-in ES cells were differentiated in stromalcell co-culture to yield cells suited for use as reprogrammable somaticcells. These cells are a model for cells isolated from a post-natalindividual for use in a somatic cell reprogramming method.

The methods were repeated with other differentiated cell types. One celltype was human fetal lung fibroblast cells, IMR-90. See, Nichols W, etal., Science 196:60-63 (1977), incorporated herein by reference as ifset forth in its entirety. IMR-90 cells are being extensivelycharacterized by the ENCODE Consortium, are readily available fromAmerican Type Culture Collection (ATCC; Manassas, Va.; Catalog No.CCL-186), and have published DNA fingerprints that allow independentconfirmation of the origin of reprogrammed clones. In addition, thesecells proliferate robustly in Eagle's Minimal Essential Medium-10% FBSfor more than twenty passages before undergoing senescence, but growslowly in human ES cell culture conditions, a difference that provides aproliferative advantage to reprogrammed clones and aids in theirselection by morphological criteria alone. Other differentiated celltypes used in the methods were human post-natal foreskin fibroblastcells (ATCC; Catalog No. CRL-2097) and human adult skin cells (ATCC;Catalog No. CRL-2106).

The cells were made receptive for transduction with a viral expressionsystem as described below. The somatic cells were transduced withpolynucleotides encoding potency-determining factors thought to beassociated with pluripotency, such that the somatic cells werereprogrammed to pluripotent cells. It is not yet determined whether allfourteen potency-determining factors provided in transduction vectorswere taken up and expressed in the somatic cells. Having identified aset of fourteen potency-determining factors, and a subset of at leasttwo of the fourteen factors, sufficient to reprogram somatic cells, theinventors provide one of ordinary skill in art the with the ability toidentify one or more specific subsets of the potency-determining factorsthat are also capable of somatic reprogramming, thereby facilitatingidentification of other subsets of such potency-determining factors.Accordingly, the methods described below facilitate the identificationof the potency-determining factors involved in reprogramming somaticcells into pluripotent cells.

It is specifically envisioned that the set of potency-determiningfactors sufficient to reprogram somatic cells can vary with the celltype of the somatic cells. It is noted that exposure to a set offourteen potency-determining factors resulted in conversion to apluripotent status in cultures of the indicated somatic cells. As shownbelow, one can identify a set of potency-determining factors sufficientto reprogram other cell types by repeating the methods described belowusing different combinations of potency-determining factors, which mayinclude some or all of the fourteen factors as well as otherpotency-determining factors. Consequently, one can produce pluripotentcell lines/populations that are substantially genetically identical to apre-existing, differentiated, somatic cell.

EXAMPLES

In the following examples, differentiated cells received vectors thatencoded various potency-determining factors. Some of the cells containedin their genome a marker gene that encodes EGFP positioned downstreamfrom the regulated Oct4 promoter, which is active only in pluripotentcells. The production of this useful tool is described in Yu et al.,supra, which demonstrated that differentiated cells can becomepluripotent via cell-cell fusion with human ES cells.

Example 1 Lentiviral Vector Packaging and Production

Transgene-expressing lentivirus vector was produced in 293FT cell lines(Invitrogen). 293T is a fast-growing, highly transfectable clonalvariant derived from transformed 293 embryonal kidney cells, whichcontains the large T antigen for high-level expression of the packagingproteins that contribute to higher viral titers. For routine maintenanceand expansion, these cells were cultured in 293FT medium (DMEM/10% FBS,2 mM L-glutamine and 0.1 mM MEM Non-Essential Amino Acids) in thepresence of 500 μg/ml geneticin. For packaging, 293FT cells werecollected by trypsinization. Following removal of trypsin bycentrifugation, these cells were aliquoted into T75 flasks (15×10⁶cells/flask, and 6 flasks per construct) in 293FT medium withoutgeneticin.

Co-transfection of lentiviral vector and two helper plasmids was carriedout with Superfect® transfection reagent (Qiagen) immediately followingcell aliquoting (lentiviral vector:MD.G:pCMVdeltaR8.9:Superfect®=5 μg:5μg:10 μg:40 μl in 400 μl of Iscove's Modified Dulbecco's Medium (IMDM)(1×)/flask incubated at room temperature for 10 minutes). The next day,the culture medium containing the transfection mixture was replaced withfresh 293FT medium supplemented with 1 mM sodium pyruvate (8 ml/flask).Lentivirus-containing supernatant was collected around 48 to 72 hoursafter transduction (˜48 ml per construct). The 293FT cell debris wasremoved from the supernatant by centrifugation at 3000 rpm (1750 g) for15 minutes at 4° C. To concentrate the lentivirus, the supernatant wasfiltered through 0.4 μM cellulose acetate (CA) membrane (Cornington, 115ml low-protein binding), and ultracentrifuged in 70 ml sterilizedbottles (Beckman, Cat#355622, polycarbonate for 45Ti rotor only) at33,000 rpm (50,000 g) for 2.5 hours at 4° C. Lentivirus along with anyremaining cell debris formed a visible pellet at the bottom of thecentrifuge tube. Following supernatant removal, PBS (˜300 μl for eachconstruct) was added to resuspend the pellet by rocking the centrifugetubes at 4° C. for 8 to 14 hours, or at room temperature for 2 hours.The remaining cell debris was removed by centrifugation at 5000 rpm(2700 g) for 5 minutes, and the resuspended lentivirus was aliquoted andstored at ˜80° C. The titer obtained generally ranged between 10⁷ to 10⁸viral particles (vp)/ml after concentration. The sequence for alentivirus (pSIN4-EF2-Stella-puro; SEQ ID NO:6, with the sequence forStella from 3604 to 4083) harboring Stella (SEQ ID NO:1) is provided inthe Sequence Listing. The same sequence was used for all otherpotency-determining factors (e.g., SEQ ID NOS: 2-5), except that thesequence for Stella (SEQ ID NO:1) was replaced with the sequence ofanother potency-determining factor.

To efficiently introduce potency-determining factors into myeloid cells,inventors modified the lentiviral expression system (FIG. 4A). Inventorsreduced the size of the original lentiviral construct (>11 kb) byremoving sequences neighboring 5′ and 3′ LTRs through serial deletionanalysis. These modifications minimized the negative effect on thepackaging efficiency. The titer obtained routinely ranged between 10⁵ to10⁶ vp/ml of supernatant, and 10⁷ to 10⁸ vp/ml after concentration(through ultracentrifugation). Restriction sites were introduced intothe backbone for convenient exchanges of the coding regions for specifictransgenes.

Example 2 Reprogramming of Myeloid Precursor Cells after LentiviralTransduction and Expression of Potency-Determining Factors

To identify genes capable of reprogramming differentiated cells back toa state of pluripotency, efficient transduction of the cells isrequired. Inventors first tested the lentiviral transduction efficiencyimmediately after Percoll® purification of a human H1 Oct4 knock-in EScells (FIG. 2).

H1.1 human ES cells (WiCell Research Institute; Madison, Wis.) weremaintained on irradiated mouse embryonic fibroblasts (MEFs) in DMEM/F12culture medium consisting of 80% Dulbecco's modified Eagle's medium (nopyruvate, high glucose formulation; Invitrogen; Carlsbad, Calif.)supplemented with 20% KnockOut serum replacer, 1% non-essential aminoacids (Gibco), 1 mM L-glutamine, 0.1 mM β-mercaptoethanol (Sigma) and 4ng/ml basic fibroblast growth factor (bFGF) (all from Invitrogen unlessotherwise noted), as previously described (see Amit et al., Dev Biol.227:271-278 (2000); and Thomson et al., Science 282:1145-1147 (1998),each of which is incorporated herein by reference as if set forth in itsentirety). Feeder-free culture on Matrigel® (BD Biosciences; Bedford,Mass.) with chemically defined TeSR™ medium (StemCell Technologies,Inc.) was carried out as described in Ludwig et al. Ludwig T, et al,Nat. Methods. 3:637-646 (2006); and Ludwig T, et al., Nat. Biotechnol.24:185-187 (2006), each of which is incorporated herein by reference asif set forth in its entirety.

The H1 Oct4 knock-in ES cell line was generated from the H1.1 human EScells according to a method described by Zwaka & Thomson. U.S. PatentPublication No. 2006/0128018 and Zwaka T & Thomson J, Nat. Biotechnol.21:319-321 (2003), each of which is incorporated herein by reference asif set forth in its entirety. Briefly, a gene targeting vector wasconstructed by inserting a cassette, an IRES-EGFP, an IRES-NEO and asimian virus polyadenylation sequence (approximately 3.2 kilobases (kb))into the 3′ untranslated region of the fifth exon of the human Oct-4(octamer-binding transcription factor 4) gene, also known as POU domain,class 5, transcription factor 1 (POU5F1). This cassette was flanked inthe 5′ direction by a 6.3 kb homologous arm and by a 1.6 kb (6.5 kb inthe alternative targeting vector) homologous arm in the 3′ region (FIG.1). The cassette was inserted at position 31392 of the Oct-4 gene (SEQID NO:2). The long arm contained a sequence from 25054-31392. The shortarm contained a sequence from 31392-32970. In an alternative targetingvector, the short arm was substituted by a longer homologous region(31392-32970 in AC006047 plus 2387-7337 in gene accession numberAC004195). Isogenic homologous DNA was obtained by long-distance,genomic PCR and subcloned. All genomic fragments and the cassette werecloned into the multiple cloning site (MCS) of a cloning vector,pBluescript® SK II (GenBank Accession Number X52328; Stratagene; LaJolla, Calif.).

For electroporation, cells were harvested with collagenase IV (1 mg/ml,Invitrogen) for 7 minutes at 37° C., washed with medium and re-suspendedin 0.5 ml culture medium (1.5−3.0×10⁷ cells). To prepare the cells forelectroporation, cells were added to 0.3 ml phosphate-buffered saline(PBS; Invitrogen) containing 40 mg linearized targeting vector DNA.Cells were then exposed to a single 320 V, 200 μF pulse at roomtemperature using a BioRad Gene Pulser® II (0.4 cm gap cuvette). Cellswere incubated for ten minutes at room temperature and were plated athigh-density on Matrigel®. G418 selection (50 mg/ml; Invitrogen) wasstarted 48 hours after electroporation. After one week, G418concentration was doubled. After three weeks, surviving colonies wereanalyzed individually by PCR using primers specific for the NEO cassetteand for the POU5F1 gene just downstream of 3′ homologous region,respectively. PCR-positive clones were re-screened by Southern blotanalysis using BamHI digested DNA and a probe outside the targetingconstruct.

The H1 Oct4 knock-in ES cell line expressed both EGFP and neomycinphosphotransferase (neo) from an endogenous Oct4 promoter/regulatoryregion using dual internal ribosome-entry sites (IRES) (FIG. 3).Expression of EGFP and neo in the H1 Oct4 knock-in ES cells indicated anactive, endogenous Oct4 promoter/regulatory region.

H1 Oct4 knock-in ES cells were maintained through co-culture with mouseOP9 bone marrow stromal cells (FIG. 2A) maintained on gelatin-coated 10cm plastic dishes (BD Biosciences) consisting of: DMEM medium(Invitrogen) supplemented with 20% non-heat-inactivated defined fetalbovine serum (FBS; HyClone Laboratories; Logan, Utah) (10 ml/dish). TheOP9 cultures were split every 4 days at a ratio of 1:7. For use in humanES cell differentiation, after OP9 cells reached confluence on thefourth day, half of the medium was changed, and the cells were culturedfor an additional four days.

For reprogramming, H1 Oct4 knock-in ES cells were differentiated intoattached cells (i.e., CD29+CD44+CD166+CD105+CD73+CD31−). Briefly, humanH1 Oct4 knock-in ES cells (p76 to 110) were added to the OP9 monolayer(1.5×10⁶/10-cm dish) in 20 ml of DMEM medium supplemented with 10% FBS(HyClone Laboratories) and 100 μM monothioglycerol (MTG; Sigma; St.Louis, Mo.). The human ES/OP9 cell co-culture was incubated for ninedays with changes of half of the medium on days 4, 6 and 8. Afterincubation, the co-culture was dispersed into individual cells bycollagenase IV treatment (1 mg/ml in DMEM medium, Invitrogen) for 20minutes at 37° C., followed by trypsin treatment (0.05% Trypsin/0.5 mMEDTA, Invitrogen) for 15 minutes at 37° C. Cells were washed twice withmedium and re-suspended at 2×10⁶/ml in DMEM medium supplemented with 10%FBS, 100 μM MTG and 100 ng/ml GM-CSF (Leukine; Berlex Laboratories Inc.;Richmond, Calif.). Cells were further cultured in flasks coated withpoly (2-hydroxyethyl methacrylate) (pHEMA; Sigma) for 10 days withchanges of half of the medium every 3 days. During adhesion-preventingpHEMA culture, cells that would otherwise be adherent formed floatingaggregates, while the cells of interest grew as individual cells insuspension. Large cell aggregates were removed by filtration through 100μM cell strainers (BD Biosciences), while small aggregates and deadcells were removed by centrifugation through 25% Percoll® (Sigma). Thedifferentiated cells recovered from the cell pellet expressed CD33, MPO,CD11b and CD11c molecules, which are characteristic for bone marrowmyeloid cells (FIG. 2B). Inventors routinely produce 6−10×10⁶differentiated cells from 1×10⁶ H1 ES cells (human H1 Oct4 knock-in EScells). See also, Yu J, et al., Science 318:1917-1920 (2007), includingthe supplemental materials available at the Science website on the WorldWide Web, incorporated herein by reference as if set forth in itsentirety.

Lentivirus encoding a potency-determining factor (MOI: 3 to 10) wasadded to the cell culture after addition of polybrene carrier at a finalconcentration of 6 μg/ml (Sigma). The lentivirus-containing medium wasreplaced with fresh medium the next day, and cells were cultured furtherin appropriate medium. Drug selection, if needed, started the third dayafter transduction. As shown in FIG. 5B, the transduction efficiency wasvery low (˜18.4% at MOI of 10). Moreover, the expression of EGFP wasbarely above background. Similar results have been obtained with routineplasmid or Epstein-Barr virus nuclear antigen (EBNA)-based plasmidtransfections (data not shown).

On the other hand, cells having high transduction efficiency wereprepared as follows. The Percoll®-purified H1 Oct4 knock-in ES cellswere allowed to differentiate further to mesenchymal-like cells for anadditional seven days in the presence of GM-CSF on Matrigel®, asdescribed above. Many cells attached to the plate during this cultureperiod. The attached cells (referred to herein below as Oct4KICD45+Acells, or simply as CD45+A cells) showed significantly highertransduction efficiency (FIG. 4C) and were used for this reprogrammingexperiment. While the cells were not CD45 at the time of theexperiments, the cells were obtained from CD45 cells. As noted elsewhereherein, cell surface markers on the attached cells were characterized asCD29⁺, CD44⁺, CD166⁺, CD105⁺, CD73⁺ and CD31⁻.

Inventors tested the hypothesis that differentiated cells could bereprogrammed to a state of pluripotency by expressingpotency-determining factors in Oct4KICD45+A cells (FIG. 3), and obtainedpromising results. Because Nanog and Oct-4 are the best characterizedpotency-determining factors, inventors examined the effect of theirover-expression in the cells.

The Oct4KICD45+A cells were first dissociated to individual cells withtrypsin and replated onto Matrigel® at ˜10⁵ cells/well of 6-well platesin TeSR™ medium. Transgene-expressing lentiviral transduction wascarried out the next day. Nanog-expressing Oct4KICD45+A cells showedsimilar morphology to that of EGFP transfected cells (FIG. 5). Nanogover-expression, however, significantly enhanced Oct4KICD45+A cellproliferation, similar to that observed in human ES cells. Followingneomycin selection for an active endogenous Oct4 promoter/regulatoryregion, no Nanog- or EGFP-transfected cells survived. Importantly, theseresults indicate that a drug-selectable population of Oct-4-expressingES cells does not persist through the culture period necessary fordifferentiation. Oct-4 expression resulted in dramatic morphologicalchanges (FIG. 5), and many of these cells survived neomycin selection.None of these cells, however, exhibited morphology typical of human EScells. The Oct4KICD45+A cells co-expressing Nanog and Oct-4 showedmorphological changes similar to those observed in cells expressingOct-4 alone. Thus, it appears that the two key potency-determiningfactors, Nanog and Oct-4, alone were not sufficient to convertdifferentiated cells to pluripotency.

Cells were analyzed using cell-sorting methods before and after exposingthe somatic cells to the factors. Adherent cells were individualized bytrypsin treatment (0.05% Trypsin/0.5 mM EDTA, Invitrogen), and fixed in2% paraformaldehyde for 20 minutes at room temperature. The cells werefiltered through a 40-μm mesh, and resuspended in FACS buffer (PBScontaining 2% FBS and 0.1% sodium azide). Cells grown in suspension werestained in the FACS buffer supplemented with 1 mM EDTA and 1% normalmouse serum (Sigma). Intracellular myeloperoxidase (MPO) staining wasperformed using Fix & Perm® reagents (Caltag Laboratories; Burlingame,Calif.). About 100 μl of cell suspension containing 5×10⁵ cells was usedin each labeling. Both primary and secondary antibody incubation (whereapplied) were carried out at room temperature for 30 minutes. Controlsamples were stained with isotype-matched control antibodies. Afterwashing, the cells were resuspended in 300-500 μl of the FACS buffer,and analyzed on a FACSCalibur flow cytometer (BDIS; San Jose, Calif.)using CellQuest™ acquisition and analysis software (BDIS). A total of20,000 events were acquired. All of the antibodies used in the flowcytometry analysis are listed in Table 1. The final data and graphs wereanalyzed and prepared using FlowJo software (Tree Star, Inc.; Ashland,Oreg.).

TABLE 1 Antibodies for flow cytometry. Clone/ ANTIGEN LABEL Product#ISOTYPE VENDOR SSEA-3 None MAB4303 ratIgM Chemicon SSEA-3 None14-8833-80 ratIgM eBioscience SSEA-4 None MAB4304 mIgG3 Chemicon SSEA-4APC FAB1435A mIgG3 R&D systems Tra-1-60 None MAB4360 mIgM ChemiconTra-1-81 None MAB4381 mIgM Chemicon CD29 PE MCA2298PE IgG AbD SerotecTra-1-85 APC FAB3195A mIgG1 R&D Systems CD140a PE 556002 mIgG2a BDPharmingen CD56 PE 340724 mIgG2b BDIS CD73 PE 550257 mIgG1 BD PharmingenCD105 PE MHCD10504 mIgG1 Caltag CD31 FITC 557508 mIgG1 BD PharmingenCD34 FITC 555821 mIgG1 BD Pharmingen BD Pharmingen (San Diego, CA) BDImmunocytometry Systems (BDIS) (San Jose, CA) Caltag Laboratories(Burlingame, CA) Chemicon International (Temecula, CA) AbD Serotec(Raleigh, NC) NA—not applicable.

To further evaluate the potency-determining factors involved inreprogramming these cells, inventors explored the transduction of poolsof ES cells enriched with various combinations of potency-determiningfactors. An exemplary pool of potency-determining factors forreprogramming myeloid precursors included the fourteenpotency-determining factors described in Table 2 below.

TABLE 2 Human ES cell-enriched genes. GENE SYMBOL UNIGENE ID ENTREZ IDACCESSION POU5F1 Hs.249184 5460 NM_002701 (Human Oct-4) Sox2 Hs.5184386657 NM_003106 Nanog Hs.329296 79923 NM_024865 FoxD3 Hs.546573 27022NM_012183 UTF1 Hs.458406 8433 NM_003577 Stella Hs.131358 359787NM_199286 Rex1 Hs.335787 132625 NM_174900 ZNF206 Hs.334515 84891NM_032805 Sox15 Hs.95582 6665 NM_006942 Mybl2 Hs.179718 4605 NM_002466Lin28 Hs.86154 79727 NM_024674 DPPA2 Hs.351113 151871 NM_138815 ESG1Hs.125331 340168 NM_001025290 Otx2 Hs.288655 5015 NM_172337

The expression of at least some of these fourteen factors in theOct4KICD45+A cells resulted in colonies with typical morphology ofpluripotent cells, such as human ES cells (FIG. 6A—left-hand photos).After neomycin selection from ˜10⁵ starting Oct4KICD45+A cells, over tencolonies having the distinct ES cell morphology initially appeared. Morethan half of these colonies were subsequently lost to differentiation,suggesting either that over-expression of one or more introduced geneshad a negative effect on the cells or that the cells continued to dependupon the foreign transgenes and gene silencing. Nevertheless, survivingcolonies expressed the endogenous Oct4 promoter-driven EGFP (FIG.7A-right-hand photos), indicating that the endogenous Oct4promoter/regulatory region was reactivated.

In this embodiment, EGFP expression occurs when the native Oct4promoter/regulatory region is active. In other words, undifferentiatedcells are identified by a green color that disappears when the cellsdifferentiate. Thus, the expression of endogenous Oct-4 in the primateES cells was selectable. These colonies also expressed Oct-4, SSEA3,SSEA4, Tra-1-60 and Tra-1-81 pluripotent cell-specific markers (FIG.6B). Similar results were obtained in reprogrammed colonies obtainedusing chemically defined TeSR™ medium.

Inventors randomly picked six colonies from two separate transfectionswith the same pool of fourteen ES cell-enriched potency-determiningfactors, and propagated five stable colonies for at least eight weeks.Thus, inventors identified a novel approach for reprogramming primatesomatic cells to become higher potency cells by administering fourteenpotency-determining factors into the somatic cells.

When these cells were exposed to other combinations ofpotency-determining factors (i.e., Sox2, c-Myc, Oct 3/4 and Klf4) usingthe lentiviral delivery system described herein, reprogramming andconversion of the cells were not observed.

Inventors used the techniques described herein to screen for subsets ofthe fourteen tested factors that are sufficient to reprogram the testedcells. Inventors' set of fourteen sufficient factors was subsequentlynarrowed to a set of six, and then four genes sufficient to reprogramthese cells (FIGS. 7A-B; described further below). The four genes shownto be sufficient in combination to yield stable pluripotent cell wereOct-4, Nanog, Sox2 and Lin28, as shown in FIG. 7B.

Example 3 Reprogramming of Mesenchymal-Like Cells with a Limited Set ofFour Potency-Determining Factors after Lentiviral Transduction

To identify a more limited set of potency-determining factors capable ofreprogramming differentiated cells back to pluripotency, theabove-identified methods were repeated with a combination of Pou5F1(Oct-4), Nanog, Sox2 and Lin28. Inventors used the techniques describedabove to screen these potency-determining factors for their ability toreprogram cells.

A different cell type was used in this example to further demonstratethe utility of the methods. The cell type was a mesenchymal-like clonalcell directly differentiated from human H1 Oct4 knock-in ES cells, asdescribed above. As used herein, “clonal” refers to a characteristic ofa population of cells derived from a common ancestor (i.e., derived froma single cell, not derived from a cell aggregate). That is, in a “clonalpopulation,” the cells display a uniform pattern of cell surface markersand morphological characteristics, as well as being substantiallygenetically identical.

Briefly, human H1 Oct4 knock-in ES cells (p76 to p110) were induced todifferentiate in co-culture with mouse OP9 bone marrow stromal cells.See, Vodyanyk M, et al., Blood 105:617-626 (2005), incorporated hereinby reference as if set forth in its entirety. Small aggregates of humanH1 Oct4 knock-in ES cells were added to OP9 cells in alpha MEMsupplemented with 10% FCS and 100 μM MTG (Sigma). On the next day(day 1) of culture, the medium was changed, and the cultures wereharvested on the days indicated below.

On day 2 of co-culture, mesodermal commitment was detected by a peakexpression of transcription factors for mesendoderm (GSC, MIXL1 and T(BRACHYURY)) and early mesoderm (EVX1, LHX1 and TBX6) with NimbleGen®(Madison, Wis.) microarrays. During days 3-5, specification of endodermand mesodermal lineages was observed. This stage was accompanied withsustained expression of genes involved in epithelial-mesenchymaltransition (EMT; SNAIL and SLUG) and cell expansion (HOXB2-3). It alsocoincided with a maximal cell proliferation rate in human H1 Oct4knock-in ES cells/OP9 co-culture.

Differentiation of specific mesendodermal lineages was observed on days5-7 of co-culture, when markers of developing endoderm (AFP andSERPINA1), mesenchymal (SOX9, RUNX2 and PPARG2) and hematoendothelial(CDH5 and GATA1) cells were detected. However, muscle-inductive factors(MYOD₁, MYF5 and MYF6) were not expressed throughout seven days ofco-culture. Moreover, neuroectoderm (SOX1 and NEFL) or trophectoderm(CGB and PLAC) markers were not detected, indicating that OP9 cellsprovided an efficient inductive environment for directed hESCdifferentiation toward the mesendodermal pathway.

Also on day 2, a single-cell suspension of the human ES cell-derivedcells was harvested by successive enzymatic treatment with collagenaseIV (Gibco-Invitrogen) at 1 mg/ml in DMEM/F12 medium for 15 minutes at37° C. and 0.05% Trypsin-0.5 mM EDTA (Gibco-Invitrogen) for 10 minutesat 37° C. Cells were washed 3 times with PBS-5% FBS, filtered through 70μM and 30 μM cell strainers (BD Labware; Bedford, Mass.) and labeledwith anti-mouse CD29-PE (AbD Serotec; Raleigh, N.C.) and anti-PEparamagnetic monoclonal antibodies (Miltenyi Biotech; Auburn, Calif.).The cell suspension was purified with magnet-activated cell sorting(MACs) by passing it through a LD magnetic column attached to aMidi-MACS separation unit (Miltenyi Biotech) to obtain a negativefraction of OP9-depleted, human H1 Oct4 knock-in ES cell-derived cells.Purity of human H1 Oct4 knock-in ES cell-derived cells was verifiedusing pan anti-human TRA-1-85 monoclonal antibodies (R&D Systems;Minneapolis, Minn.).

Purified human H1 Oct4 knock-in ES cell-derived cells were plated atdensity of 2×10⁴ cells/ml on semisolid, serum-free medium composed ofStemLine™ serum-free medium (Sigma) supplemented with 5-100 ng/ml basicfibroblast growth factor (bFGF; PeproTech; Rocky Hill, N.J.) and 1%methylcellulose (StemCell Technologies, Inc.) with or without 10-20ng/ml PDGF-BB (PeproTech). PDGF-BB improved growth of mesenchymal cells,but was not essential for colony formation. After 14-21 days of culture,large, compact mesenchymal colonies formed, resembling embryoid bodies(EBs). Mesenchymal colonies were detected on day 7; however, 14-21 dayswere required to reveal actively growing colonies.

Individual mesenchymal colonies were transferred to wells of a collagen-or fibronectin-coated, 96-well plate pre-filled with 0.2 ml/wellStemLine® serum-free medium supplemented with 5-100 ng/ml bFGF. After3-4 days of culture, adherent cells from individual wells were harvestedby trypsin treatment and expanded on collagen- or fibronectin-coateddishes in StemLine® serum-free medium with 5-100 ng/ml bFGF.

Transgene-expressing lentiviral transduction was then carried out asdescribed above. Inventors tested the hypothesis that differentiatedmesenchymal-like cells could be reprogrammed to a state of pluripotencyby expressing a limited set of potency-determining factors (e.g., Oct-4,Nanog, Sox2 and Lin28). The expression of at least these fourpotency-determining factors resulted in colonies having cells withtypical morphology of pluripotent cells, such as human ES cells (FIG.7B; dark gray bars). As shown in FIG. 7B, the greatest number ofcolonies having cells with typical morphology of pluripotent cells wasobtained using the full complement of Oct-4, Nanog, Sox2 and Lin28.However, when one of Oct-4, Nanog, Sox2 or Lin28 was absent, the numberof ES-like colonies was significantly attenuated (e.g., Nanog or Lin28)or absent (e.g., Oct-4 or Sox2).

In this embodiment, EGFP expression occurred when the native Oct4promoter/regulatory region was active. In other words, undifferentiatedcells were identified by a green color that was absent fromdifferentiated cells. Thus, the expression of endogenous Oct-4 in thecells was selectable. Reprogrammed colonies also expressed Oct-4, SSEA3,SSEA4, Tra-1-60 and Tra-1-81 pluripotent cell-specific markers (data notshown).

Inventors randomly picked six colonies from two separate transfectionswith the same pool of fourteen ES cell-enriched potency-determiningfactors, and propagated five stable colonies for at least eight weeks.Thus, inventors identified a novel approach for reprogramming primatesomatic cells to become higher potency cells by administering fourpotency-determining factors into the somatic cells.

When these cells were exposed to other combinations ofpotency-determining factors (i.e., Sox2, c-Myc, Oct 3/4 and Klf4) usingthe lentiviral delivery system described herein, reprogramming andconversion of the cells were not observed.

Example 4 Reprogramming of Mesenchymal-Like Cells with a Limited Set ofTwo Potency-Determining Factors after Lentiviral Transduction

To identify an even more limited set of potency-determining factorscapable of reprogramming differentiated cells back to pluripotency, theabove-identified methods were repeated in the mesenchymal-like cells ofExample 3 with a combination of two of the following fourpotency-determining factors: Oct-4, Nanog, Sox2 and Lin28. Inventorsused the techniques described above to screen these potency-determiningfactors for their ability to reprogram cells.

Transgene-expressing lentiviral transduction was then carried out asdescribed above. Inventors tested the hypothesis that differentiatedmesenchymal-like cells could be reprogrammed to a state of pluripotencyby expressing fewer than four potency-determining factors. Theexpression of at least Oct-4 and Sox2 (FIG. 7C) resulted in colonieshaving cells with typical morphology of pluripotent cells, such as humanES cells. Nanog and Lin28, singly and in combination, had a beneficialeffect in clone recovery by improving reprogramming efficiency in humanES cell-derived mesenchymal cells to a state of pluripotency, but wereessential neither for the initial appearance of reprogrammed cells norfor the expansion of reprogrammed cells.

Example 5 Reprogramming of a Differentiated Cells after LentiviralTransduction and Expression of Four Potency-Determining Factors

To further demonstrate the utility of the limited set ofpotency-determining factors in reprogramming differentiated cells backto pluripotency, the above-identified methods were repeated with ATCCCatalog No. CCL-186 (IMR-90; ATCC), which are human fetal lungfibroblast cells (see also, Birney E, et al., Nature 447:799-816(2007)).

Transgene-expressing lentiviral transduction was carried out asdescribed above. That is, IMR-90 cells (0.9×10⁶/well), were transducedwith a combination of Oct-4, Sox2, Nanog and Lin28. Inventors tested thehypothesis that differentiated fibroblast cells could be reprogrammed toa state of pluripotency by expressing a limited set ofpotency-determining factors (e.g., Oct-4, Sox2, Nanog and Lin28).Following transduction, cells were transferred to three 10-cm dishesseeded with irradiated mouse embryonic fibroblasts (MEFs). By day 12post-transduction, small colonies with human ES cell morphology becamevisible. On day 20 post-transduction, a total of 198 colonies werevisible on 3 plates. Forty-one of the colonies were picked, thirty-fiveof which were successfully expanded for an additional three weeks. Sixof these colonies were then selected for continued expansion andanalysis, and the other twenty-nine were frozen.

The introduction of at least Oct-4, Sox2, Nanog and Lin28 resulted incolonies with typical morphology of pluripotent cells like human EScells that had a normal karyotype. Cells from each colony likewiseexpressed telomerase activity and expressed human ES cell-specificsurface antigens (i.e., SSEA-3, SSEA-4, Tra-1-60 and Tra1-81). For eachof the colonies, the expression of endogenous OCT4 and NANOG was atlevels similar to that of pluripotent cells, although the exogenousexpression of these genes did vary. Moreover, EB and teratoma formationdemonstrated that the reprogrammed cells had a developmental potentialto give rise to differentiated derivatives of all three primary germlayers.

DNA fingerprint analysis confirmed that these colonies were derived fromIMR-90 cells and that they were not derived from human ES cells lines(e.g., H1, H7, H9, H13 and H14).

Similar to the data obtained with differentiated mesenchymal cells, thegreatest number of colonies having cells with typical morphology ofpluripotent cells, such as human ES cells was obtained using the fullcomplement of Oct-4, Nanog, Sox2 or Lin28. However, when Oct-4, Nanog,Sox2 or Lin28 were absent, the number of ES-like colonies wassignificantly attenuated (e.g., Nanog or Lin28) or absent (e.g., Oct-4or Sox2).

The colonies selected for expansion and detailed characterizationproliferated for at least twelve weeks and retained typicalcharacteristics of normal pluripotent cells, even though no selectionfor the activation of a pluripotency-specific gene was applied duringreprogramming.

Reprogrammed cells were identified based on morphology alone (i.e.,having a compact colony with high nucleus to cytoplasm ratio andprominent nucleolus). Reprogrammed cells also expressed Oct-4, SSEA3,SSEA4, Tra-1-60 and Tra-1-81 pluripotent cell-specific markers.

Example 6 Reprogramming of Differentiated Cells after LentiviralTransduction and Expression of Three Potency-Determining Factors

To further demonstrate the utility of the limited set ofpotency-determining factors in reprogramming differentiated cells backto pluripotency, the above-identified methods were repeated with theIMR-90 cells, described above. In this set of experiments, fewerpotency-determining factors were used than in Example 5.

Transgene-expressing lentiviral transduction was carried out asdescribed above. IMR-90 cells were transduced with a combination ofthree of the following: Oct-4, Sox2, Nanog and Lin28. Inventors testedthe hypothesis that differentiated fibroblast cells could bereprogrammed to a state of pluripotency by expressing the even morelimited set of potency-determining factors. The expression of at leastthree factors resulted in colonies with typical morphology ofpluripotent cells like human ES cells. Reprogrammed colonies havingcells with typical morphology of pluripotent cells were obtained usingthe full complement of Oct-4, Sox2 and Nanog with or without Lin28.Therefore, the presence or absence of Lin28 did not affectreprogramming. However, when any of Oct-4, Nanog or Sox2 was absent, thenumber of reprogrammed colonies was significantly attenuated or absent.

To examine for the presence of Oct-4, Sox2, Nanog and Lin28 provirus inthe reprogrammed cells, PCR with transgene-specific primer pairs (see,Table 3; one gene-specific primer and one lentiviral vector-specificprimer) was carried out using genomic DNA from IMR-90 clones astemplate. The reactions employed the pfx DNA polymerase (Invitrogen,amplification buffer was used at 2×, and enhancer solution was used at3×), and the following conditions: initial denaturation for 1 minute at95° C.; 35 cycles of 94° C. for 30 seconds, 55° C. for 30 seconds, 68°C. for 2 minutes; and followed by 68° C. for 7 minutes. PCR analysis forthe transgenes showed that either all four transgenes or threetransgenes (i.e., Oct-4, Sox2 and Nanog) integrated into the pluripotentcells following exposure to transgene-expressing lentivirus vectors.

TABLE 3 Primer sets for assessing provirus integration. Size Genes (bp)Sequences (5′ to 3′) OCT4 656 OCT4-F1 CAGTGCCCGAAACCCACAC (SEQ ID NO: 7)SP3 AGAGGAACTGCTTCCTTCACGACA (SEQ ID NO: 8) NANOG 732 NANOG-F2CAGAAGGCCTCAGCACCTAC (SEQ ID NO: 9) SP3 AGAGGAACTGCTTCCTTCACGACA(SEQ ID NO: 8) SOX2 467 SOX2-F1 TACCTCTTCCTCCCACTCCA (SEQ ID NO: 10) SP3AGAGGAACTGCTTCCTTCACGACA (SEQ ID NO: 8) LIN28 518 LIN28-F1AAGCGCAGATCAAAAGGAGA (SEQ ID NO: 11) SP3 AGAGGAACTGCTTCCTTCACGACA(SEQ ID NO: 8) OCT4endo 113 OCT4-F2 AGTTTGTGCCAGGGTTTTTG (SEQ ID NO: 12)OCT4-R2 ACTTCACCTTCCCTCCAACC (SEQ ID NO: 13)

Reprogrammed cells were identified based on morphology alone (i.e.,having a compact colony with high nucleus to cytoplasm ratio andprominent nucleolus). Reprogrammed cells also expressed Oct-4, SSEA3,SSEA4, Tra-1-60 and Tra-1-81 pluripotent cell-specific markers.

Example 7 Reprogramming of Differentiated Cells after LentiviralTransduction and Expression of Three Potency-Determining Factors

To further demonstrate the utility of the limited set ofpotency-determining factors in reprogramming differentiated cells topluripotency, the above-identified methods were repeated with ATCCCatalog No. CRL-2097 (ATCC), which are human post-natal foreskinfibroblast cells.

Transgene-expressing lentiviral transduction was carried out asdescribed above. Post-natal fibroblast cells (0.6×10⁶/well) weretransduced with a combination of Oct-4, Sox2, Nanog and Lin28. Inventorstested the hypothesis that differentiated, post-natal, fibroblast cellscould be reprogrammed to a state of pluripotency by expressing a limitedset of potency-determining factors and obtained promising results.Following transduction, cells were transferred to three 10-cm dishesseeded with irradiated MEFs. By day 15 post-transduction, small colonieswith pluripotent cell morphology became visible. On day 20post-transduction, a total of 57 colonies were visible on the plates.Twenty-nine of the colonies were picked, twenty-seven of which weresuccessfully expanded for an additional three weeks. Four of thesecolonies were then selected for continued expansion and analysis, andthe other twenty-three were frozen.

The expression of Oct-4, Sox2, Nanog and Lin28 resulted in colonieshaving cells with typical morphology of pluripotent cells, such as humanES cells, and a normal karyotype. Reprogrammed colonies likewiseexpressed telomerase activity and expressed pluripotent cell-specificmarkers (i.e., SSEA-3, SSEA-4, Tra-1-60 and Tra1-81). For each,endogenous OCT4 and NANOG was expressed at levels similar to thatobserved in human pluripotent cells, although the exogenous expressionof these genes varied. Moreover, EB and teratoma formation demonstratedthat the reprogrammed cells had a developmental potential to give riseto differentiated derivatives of all three primary germ layers. However,in contrast to the iPS cells obtained from IMR-90 cells, iPS cellsderived from CRL-2097 cells showed a variation in the lineages apparentin teratomas examined at five weeks. Two of the iPS cell colonies showedneural differentiation; whereas the other two colonies showed multiplefoci of columnar epithelial cells, reminiscent of primitive ectoderm.

DNA fingerprint analysis confirmed that these colonies were derived fromthe original cell line and confirmed that they were not derived fromhuman ES cells lines (e.g., H1, H7, H9, H13 and H14).

Similar to the data obtained after transduction of differentiatedmesenchymal cells, the greatest number of colonies having cells withtypical morphology of human pluripotent cells were obtained using thefull complement of Oct-4, Sox2, Nanog and Lin28. Interestingly, one cellline lacked Lin28, confirming that Lin28 was not essential forreprogramming somatic cells.

The colonies selected for expansion and detailed characterizationproliferated for at least twelve weeks and retained typicalcharacteristics of normal human pluripotent cells, even though noselection for the activation of a pluripotency-specific gene was appliedduring reprogramming.

Reprogrammed cells were identified based on morphology alone (i.e.,having a compact colony with high nucleus to cytoplasm ratio andprominent nucleolus). Reprogrammed cells also expressed Oct-4, SSEA3,SSEA4, Tra-1-60 and Tra-1-81 pluripotent cell-specific markers.

When these cells were exposed to other combinations of factors (i.e.,Sox2, c-Myc, Oct 3/4 and Klf4) using the lentiviral delivery systemdescribed herein, reprogramming and conversion of the cells were notobserved.

Example 8 Reprogramming of Differentiated Cells after LentiviralTransduction and Expression of Four Potency-Determining Factors

To further demonstrate the utility of the limited set ofpotency-determining factors in reprogramming differentiated cells topluripotency, the above-identified methods were repeated with ATCCCatalog No. CRL-2106 (SK46; ATCC), which are human adult skin cells.

Transgene-expressing lentiviral transduction was carried out asdescribed above. That is, skin cells (2.0×10⁵/well) were transduced witha combination of Oct-4, Sox2, Nanog and Lin28. Inventors tested thehypothesis that adult skin cells could be reprogrammed to a state ofpluripotency by expressing a limited set of potency-determining factorsand obtained promising results. Following transduction, cells weretransferred to three 10-cm dishes seeded with irradiated mouse embryonicfibroblasts (MEFs). After 10 days in human ES cell culture medium humanES cell culture medium conditioned with irradiated MEFs was used tosupport cell growth. By day 18 post-transduction, small colonies withpluripotent cell morphology became visible.

The expression of Oct-4, Sox2, Nanog and Lin28 resulted in colonieshaving cells with typical morphology of pluripotent cells (see, FIG.8A), such as human ES cells (i.e., having a compact colony with highnucleus to cytoplasm ratio and prominent nucleolus). As shown in FIG.8B, the reprogrammed cells also expressed cell surface markers typicalof pluripotent cells; SK46 cells (control), however, did not. However,the reprogrammed colonies from adult skin cells appeared later than thecells in Example 7 and had a lower reprogramming efficiency than thecells in Example 7.

Example 9 Increasing Reprogramming Efficiency by LinkingPotency-Determining Factors on a Single Construct

To increase the reprogramming efficiency, the above-identified methodswere repeated using the construct shown in FIG. 4A; however, eitherOct-4 or Sox2 were inserted in the transgene section, and Sox2optionally replaced the puromycin resistance gene. The constructs werethen expressed either in 293FT cells or in OCT4 knock-in human H1 EScells (p6).

Transgene-expressing lentiviral transduction was carried out asdescribed above. That is, 293FT cells or mesenchymal cells (˜2×10⁵cells/well of 6-well plate, seeded overnight) were transduced withvarious transgene combinations. Cells were transferred to 10 cm MEF dish(1 well of 6-well plate to 1×10 cm MEF dish) following the overnightincubation with lentivirus. Geneticin selection (50 μg/ml) for anactive, endogenous, OCT4 promoter was carried out between day 11 to 15post transduction. iPS colonies were counted on day 16.

FIG. 9A demonstrates that Oct-4 and Sox2 expression occurred in 293FTcells following transfection (see, e.g., lanes 1-3). In FIGS. 9A-B,pSin4-EF2-Oct4-IRES1-Sox2 is abbreviated as OS-IRES1;pSin4-EF2-Oct4-IRES2-Sox2 is abbreviated as OS-IRES2;pSin4-EF2-Oct4-F2A-Sox2 is abbreviated as OS-F2A;pSin4-EF2-Oct4-IRES1-puro is abbreviated as O; andpSin4-EF2-Sox2-IRES1-puro is abbreviated as S.

FIG. 9B shows that reprogramming efficiency increased in mesenchymalcells derived from OCT4 knock-in human H1 ES cells (p6) when Oct-4 andSox2 were provided on the same construct (IRES1 is a very low-efficiencyinternal ribosome entry site; whereas IRES2 is a high-efficiencyinternal ribosome entry site). OS-IRES2+N+L (the high-efficiency IRES)showed an approximate four fold increase in reprogramming efficiencywhen compared to O+S, O+S+N+L or OS-IRES1 (the low-efficiency IRES)+N+L.Therefore, providing the potency-determining factors in one constructthat provides for approximately equal expression levels of each canimprove reprogramming efficiency.

It is understood that certain adaptations of the invention described inthis disclosure are a matter of routine optimization for those skilledin the art, and can be implemented without departing from the spirit ofthe invention, or the scope of the appended claims.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. It is understood, however, that examples and embodimentsof the present invention set forth above are illustrative and notintended to confine the invention. The invention embraces all modifiedforms of the examples and embodiments as come within the scope of thefollowing claims.

We claim:
 1. A method of reprogramming primate somatic cells, the methodcomprising the steps of: introducing a plurality of potency-determiningfactors into the primate somatic cells under conditions sufficient toreprogram the cells, wherein the potency-determining factors compriseOct-4 and Sox2, and do not comprise c-Myc and Klf4; and culturing theprimate somatic cells under embryonic stem cell culture conditions toobtain pluripotent reprogrammed cells.
 2. The method of claim 1, whereinthe primate somatic cells are obtained from a post-natal individual. 3.The method of claim 1, wherein the primate somatic cells are obtained byin vitro differentiation of a stem cell.
 4. The method of claim 1,wherein the potency-determining factors are introduced to the somaticcells as a reprogramming sequence in which a nucleic acid sequenceencoding the potency-determining factor is operably linked to aheterologous promoter.
 5. The method of claim 1, wherein thepotency-determining factors are Oct-4, Sox2, and at least one of Nanogand Lin28.
 6. The method of claim 1, wherein the potency-determiningfactors are Oct-4 and Sox2.
 7. The method of claim 1, wherein thereprogrammed cells (i) express a cell marker selected from the groupconsisting of Oct-4, SSEA3, SSEA4, Tra-1-60 and Tra-1-81; (ii) exhibitmorphology characteristic of pluripotent cells; and (iii) form teratomaswhen introduced into an immunocompromised animal.
 8. A method ofreprogramming primate somatic cells, the method comprising the steps of:introducing a plurality of potency-determining factors into the primatesomatic cells under conditions sufficient to reprogram the cells,wherein the potency-determining factors comprise Oct-4, Sox2, Nanog, andLin28; and culturing the primate somatic cells under embryonic stem cellculture conditions to obtain reprogrammed cells having a higher potencylevel than the primate somatic cells.